A Single-Laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

BACKGROUND: Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains has caused a major health care burden worldwide. Cronobacter is a group of Gram-negative bacteria that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp., and Vibrio spp. is crucial for the source tracking of contaminated food, as well as to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. OBJECTIVE: This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like isolates of public health importance. METHOD: A total of 226 isolates recovered from various food, environmental surveillance samples, and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates, whole genome sequencing (WGS) and DNA sequencing of 16S rRNA partial region were also performed for species identification. RESULTS: The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system were comparable to data obtained by the VITEK MS system. CONCLUSIONS: The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates and bacterial isolates from additional sources, in most cases. HIGHLIGHTS: The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

Draft Genome Sequences of 14 Fluoroquinolone-Resistant Escherichia coli Isolates from Imported Shrimp.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA, parC, and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Draft Genome Sequences of Sixteen Fluoroquinolone-Resistant Extraintestinal Escherichia coli Isolates from Human Patients.

We report here the draft genome sequences of 16 fluoroquinolone-resistant extraintestinal Escherichia coli isolates from human patients. These isolates had high MICs (32 to 256 µg/mL) for ciprofloxacin and contained point mutations in the quinolone resistance-determining region (QRDR) of both gyrA and parC that confer resistance to fluoroquinolone. The whole-genome sequence data provide a better understanding of the fluoroquinolone resistance mechanisms in these isolates and would be beneficial in source tracking these pathogens during pandemic outbreaks.

Whole-Genome Sequences of Nine Hospital-Associated Methicillin-Susceptible Staphylococcus aureus Strains.

We present the draft genome sequences of nine hospital-associated methicillin-susceptible Staphylococcus aureus (HA-MSSA) strains. All strains were from Minnesota (eight from blood and one from bone), harbored various virulence genes, and showed diverse multilocus sequence typing and spa types.

Draft Genome Sequences of 27 Hospital-Associated Methicillin-Resistant Staphylococcus aureus Strains Isolated in Minnesota.

Infections caused by hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains have higher morbidity and mortality rates and require longer hospital stays than do those caused by hospital-associated methicillin-sensitive Staphylococcus aureus strains. To gain insight into their genomic makeup, antimicrobial resistance, biofilm formation, and virulence potentials, here we present the draft whole-genome sequences of 27 HA-MRSA strains isolated in Minnesota.

Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation.

Draft Genome Sequences of Eight Community-Associated Methicillin-Resistant Staphylococcus aureus Strains.

Here, we report the draft genome sequences of eight community-associated methicillin-resistant Staphylococcus aureus strains that were resistant to cefoxitin, ampicillin, and erythromycin. Three isolates, i.e., CAR1, CAR2, and CAR8, were sequence type 8 (ST8) with staphylococcal cassette chromosome mec (SCCmec) type IVa and were Panton-Valentine leukocidin (PVL) positive, which has been known as a predominant clone in the United States.

Genotypic Characterization of Clinical Isolates of Staphylococcus aureus from Pakistan.

In this study, we compared pulsed-field gel electrophoretic (PFGE), multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec), spa typing, and virulence gene profiles of 19 Panton-Valentine leucocidin (PVL)-positive, multidrug-, and methicillin-resistant clinical Staphylococcus aureus (MRSA) isolates obtained from a hospital intensive care unit in Pakistan. The isolates exhibited 10 pulsotypes, contained eight adhesin genes (bbp, clfA, clfB, cna, fnbA, fnbB, map-eap, and spa), 10 toxin genes (hla, hlb, hld, hlg, pvl, sed, see, seg, seh, and tst), and two other virulence genes (cfb, v8) that were commonly present in all isolates. The spa-typing indicated seven known spa types (t030, t064, t138, t314, t987, t1509, and t5414) and three novel spa types. MLST analysis indicated eight ST types (ST8, ST15, ST30, ST239, ST291, ST503, ST772, and ST1413). All isolates belonged to the agr group 1. Most of the isolates possessed SCCmec type III, but some isolates had it in combination with types SCCmec IV and V. The presence of multidrug-resistant MRSA isolates in Pakistan indicates poor hygienic conditions, overuse of antibiotics, and a lack of rational antibiotic therapy that have led to the evolution and development of hypervirulent MRSA clones. The study warrants development of a robust epidemiological screening program and adoption of effective measures to stop their spread in hospitals and the community.

Large-scale genomic analysis reveals the genetic cost of chicken domestication.

BACKGROUND: Species domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication. RESULTS: We analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts (average π, 0.00326 vs. 0.00316), the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the "cost of domestication" hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes. CONCLUSION: This study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.

Author Correction: 863 genomes reveal the origin and domestication of chicken.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

863 genomes reveal the origin and domestication of chicken.

Despite the substantial role that chickens have played in human societies across the world, both the geographic and temporal origins of their domestication remain controversial. To address this issue, we analyzed 863 genomes from a worldwide sampling of chickens and representatives of all four species of wild jungle fowl and each of the five subspecies of red jungle fowl (RJF). Our study suggests that domestic chickens were initially derived from the RJF subspecies Gallus gallus spadiceus whose present-day distribution is predominantly in southwestern China, northern Thailand and Myanmar. Following their domestication, chickens were translocated across Southeast and South Asia where they interbred locally with both RJF subspecies and other jungle fowl species. In addition, our results show that the White Leghorn chicken breed possesses a mosaic of divergent ancestries inherited from other subspecies of RJF. Despite the strong episodic gene flow from geographically divergent lineages of jungle fowls, our analyses show that domestic chickens undergo genetic adaptations that underlie their unique behavioral, morphological and reproductive traits. Our study provides novel insights into the evolutionary history of domestic chickens and a valuable resource to facilitate ongoing genetic and functional investigations of the world's most numerous domestic animal.

Draft Genome Sequences of Two Methicillin-Resistant Clinical Staphylococcus aureus Isolates.

Here, we report the draft genome sequences of two methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, hospital-associated perirectal isolate 32S (ST 239) from a colitis tracheostomy patient and community-associated MRSA isolate 42S (ST 772) from a hepatic-splenomegaly patient in Rawalpindi, Pakistan.

Genomic Sequence of a Clinical Vancomycin-Resistant Reference Strain, Enterococcus faecalis ATCC 51299.

In this paper, we present a draft genome sequence of a quality control reference strain, Enterococcus faecalis ATCC 51299 (multilocus sequencing type [MLST] ST6), which is sensitive to teicoplanin but resistant to vancomycin. It is used in an agar screening test for streptomycin, gentamicin, and vancomycin resistance and the resistance marker vanB.

Draft Genome Sequence of Multidrug-Resistant Enterococcus faecium Clinical Isolate VRE3, with a Sequence Type 16 Pattern and Novel Structural Arrangement of Tn1546.

Multidrug-resistant Enterococcus faecium has emerged as a nosocomial pathogen that may infect the body at various sites, including the gastrointestinal tract, and has serious implications in human health and disease. Here, we present the draft genome sequence of clinical strain VRE3, which exhibited a sequence type 16 (ST16) pattern and carried truncated Tn1546, a mobile genetic element encoding a high level of vancomycin resistance.

Characterisation of novel mutations involved in quinolone resistance in Escherichia coli isolated from imported shrimp.

Fifty-five nalidixic acid-resistant Escherichia coli strains were isolated from imported shrimp. Purified PCR amplicons of gyrA, gyrB, parC and parE from the template DNA of all isolates were sequenced and analysed for point mutations that confer resistance to nalidixic acid and ciprofloxacin. Point mutations in the quinolone resistance-determining regions (QRDRs) of GyrA at positions 68, 83 and 87 and in ParC at positions 80 and 84 as well as in the non-QRDR of GyrA at positions 112, 127, 128 and 154 along with point mutations in parE at position 476 conferred resistance to these antibiotics. Computational modelling and analysis of the different point mutations and their role in the enhanced resistance to these antibiotics indicated that only mutation at codons 83 (Ser→Ile) and 87 (Asp→Asn) played a vital role in increasing the minimum inhibitory concentration (MIC) to these drugs compared with other mutations. Ethidium bromide experiments indicated higher efflux pump activities in quinolone-resistant E. coli strains compared with their quinolone-sensitive counterparts. Class 1 integrons measuring 0.7-2.3kb were amplified and sequenced from the template DNA of the isolates. Sequence analysis of the 2.0kb and 1.7kb integrons indicated the presence of resistance determinants for trimethoprim (dfrA12 and dfrA17) and aminoglycosides (aadA2 and aadA5). These results indicate that use of nalidixic acid, ciprofloxacin and other antibiotics in shrimp aquaculture ponds may select E. coli resistant to these antibiotics and that imported shrimp is a reservoir of multiple antibiotic-resistant E. coli.

Bone morphogenetic protein 2: a potential new player in the pathogenesis of diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. Vision loss in DR principally occurs due to breakdown of the blood-retinal barrier (BRB), leading to macular edema, retinal detachment and inner retinal and vitreous hemorrhage. Several growth factors have been shown to play crucial role in the development of these vascular changes; however, the cellular and molecular mechanisms of DR are not yet fully revealed. In the current study we investigated the role of bone morphogenetic protein-2 (BMP2) in DR. We examined the changes in the protein levels of BMP2 in human vitreous and retina in addition to the mouse retina of streptozotocin-induced diabetes. To detect the source of BMP2 during diabetes, human retinal endothelial cells (hRECs) were subjected to high glucose (HG) for 5 days and levels of BMP2 protein were analyzed in conditioned media of these cells relative to control. We also evaluated the effect of BMP2 on the levels of VEGF in cultured rat Müller cells (rMC1). In addition, we tested the pro-inflammatory effects of BMP2 by examining its effect on leukocyte adhesion to cultured hRECs, and levels of adhesion molecules and cytokines production. Finally, the effect of different concentrations of BMP2 on permeability of confluent monolayer of hRECs was evaluated using FITC-Dextran flux permeability assay and by measuring Transcellular Electrical Resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). Our results show, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas in addition to its detection in vitreous of patients with proliferative DR (72 ± 7 pg/ml). In vitro, hRECs showed upregulation of BMP2 in HG conditions suggesting that these cells are a potential source of BMP2 in diabetic conditions. Furthermore, BMP2 induced VEGF secretion by Müller cells in-vitro; and showed a dose response in increasing permeability of cultured hRECs. Meanwhile, BMP2 pro-inflammatory effects were recognized by its ability to induce leukocyte adhesion to the hRECs, intercellular adhesion molecule-1 (ICAM-1) and upregulation of interleukin-6 and 8 (IL-6 and IL-8). These results show that BMP2 could be a contributing growth factor to the development of microvascular dysfunction during DR via enhancing both pro-angiogenic and inflammatory pathways. Our findings suggest BMP2 as a potential therapeutic target to prevent/treat DR.

Molecular characterization of fluoroquinolone-resistant Aeromonas spp. isolated from imported shrimp.

Sixty-three nalidixic acid-resistant Aeromonas sp. isolates were obtained from imported shrimp. Phylogenetic analysis of gyrB sequences indicated that 18 were A. enteropelogenes, 26 were A. caviae, and 19 were A. sobria. Double missense mutations in the quinolone resistance-determining region (QRDR) of gyrA at codon 83 (Ser→Val/Ile) and codon 92 (Leu→Met) coupled with a point mutation of parC at codon 80 (Ser→Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of A. enteropelogenes and A. caviae strains harbored toxin genes, whereas only a few A. sobria strains harbored these genes. The fluoroquinolone-resistant Aeromonas spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent Aeromonas spp. to rat epithelial cells.

Isolation and characterization of multidrug-resistant Klebsiella spp. isolated from shrimp imported from Thailand.

A study was undertaken to isolate and characterize tetracycline and nalidixic acid-resistant Klebsiella spp. in farm-raised, imported shrimp sold in the United States. Sixty-seven multiple antibiotic-resistant Klebsiella spp. strains were isolated from imported shrimp samples. Using morphological and biochemical methods, fifty-three strains were tentatively identified as Klebsiella pneumoniae and fourteen as K. oxytoca. Although all isolates were resistant to tetracycline, only 8 were resistant to nalidixic acid. These 8 isolates were further screened by PCR for quinolone resistance genes (qnrA, B, S, gyrA, B and parC). PCR protocols failed to amplify any qnr genes. The purified PCR amplicons of gyrA, gyrB and parC were sequenced and analyzed for point mutations that confer resistance to fluoroquinolone antibiotics. Analysis of the sequences of the gyrA amplicons from nalidixic acid-resistant Klebsiella spp. indicated two point mutations in gyrA at positions 83 (Ser→Phe) and 87 (Asp→Ala). Sequence analysis of the parC amplicons indicated an amino acid change at position 80 (Ser→Ile). No mutations were detected in gyrB. Template DNA from all isolates was screened for tetracycline resistance genes (tetA-E). Oligonucleotide primers specifically targeting a 305-bp region of tetB and a 477-bp region of tetD successfully amplified sequences from 91.0 and 44.0% of the isolates, respectively. None of the isolates contained tetA, tetC or tetE genes. Plasmids (2.0-16.0kb) were found in 23 of the 67 isolates. XbaI-PFGE identified 32 distinct macro restriction patterns (mrps) among the 61 multiple drug-resistant Klebsiella spp. that were typable. Our results indicate that imported shrimp is a reservoir for multidrug resistant Klebsiella spp. and potential health risks posed by such strains should not be underestimated.

Lysozyme as a barrier to growth of Bacillus anthracis strain Sterne in liquid egg white, milk and beef.

In this study, we investigated the role of lysozyme on the viability of Bacillus cereus, Bacillus subtilis, Bacillus pumilus and Bacillus anthracis (Sterne) in egg white (EW), ground beef and milk. At 35 °C in EW, growth rates (GR) for B. cereus, B. subtilis, B. pumilus and B. anthracis were 0.005, -0.018, -0.028 and -0.029 OD(600)/h, respectively. Heat-treating EW at 55 and 60 °C reduced the inactivating effect of EW by 3.1 and 10.5-fold, respectively. Addition of lysozyme (2 mg/ml) to 60 °C-treated EW increased the inactivation rate 5.76-fold, indicating involvement of lysozyme in B. anthracis inactivation. B. anthracis inactivation was influenced by pH, as shown by a progressive increase in inactivation rate from 0.25 to -4.42 logs CFU/h over a pH range of 6.0-8.5. Adding 2 mg/ml lysozyme to milk and ground beef also suppressed the growth of B. anthracis 3.3 and 6.5-fold, respectively. These data indicate that lysozyme, as a natural component of EW or potential additive in other foods, could reduce biothreat risks presented by bioterror agents.

Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant bacteria: Comparative analysis of real-time multiplex PCR assays using EvaGreen(®) and SYBR(®) Green I dyes.

We have developed multiplex real-time PCR assays that utilize DNA-intercalating dyes, SYBR Green I (SG) and EvaGreen (EG), with two primer sets (set 1=qacEδ1, tetA and aacA-aphD; set 2=tetA, marA, and floR) to simultaneously amplify the qacEδ1, tetA, aacA-aphD, marA, and floR genes. Validity of the multiplex PCR assays was confirmed by testing 83 bacterial isolates, including Staphylococcus aureus (28 isolates), Enterococcus spp. (17 isolates), Salmonella enterica serovar Typhimurium (8 isolates), Citrobacter spp. (9 isolates), Escherichia coli (14 isolates) and Aeromonas veronii (7 isolates), and performing sequence analysis of representative PCR products. Agarose gel analysis revealed the presence of correct size PCR products, and the differences in their thermal melting (T(m)) curves were used to distinguish various PCR products. Although T(m) peaks of different amplicons after EG-based singleplex and multiplex PCR assays were resolved nicely, only one or two peaks were seen for SG-bound amplicons. EG-based multiplex real-time PCR assays provided better peak resolution. There was a good correlation with a better linear relationship between the C(t) and log input DNA concentration for the set 1 and set 2 genes in EG-based assays (R(EG)(2)=0.9813and0.9803) than in SG-based assays (R(SG)(2)=0.5276and0.6255). The sensitivities of detection were 2.5-25fg and 25-250fg of template DNA in EG and SG-based singleplex and multiplex PCR assays, respectively. The assays, which could be completed in less than 45min, offer sensitive and rapid detection of qacEδ1, aacA-aphD, marA, floR, and tetA genes from a diverse group of multiple antibiotic-resistant bacterial strains.